Method of breeding of yeast on solutions, containing lactose, and ground cereal products and/or other sugar and polysaccharide products

ABSTRACT

A method of breeding yeast on residual solution containing lactose from dairies, and ground cereals, leguminous products and/or other raw materials, containing sugars and polysaccharides, ammonia being added as the sole inorganic substance.

This is a continuation of application Ser. No. 858,891, filed Dec. 8,1971, now abandoned.

The present invention relates to a method of breeding of yeast onresidual solutions containing lactose from dairies, and ground cereals,leguminous products and/or other raw materials, containing sugars andpolysaccharides, ammonia being added as the sole inorganic substance.

Residual solutions, containing lactose, from dairies are difficult toutilize, as large solution volumes with a low solids content must beworked up. A complete utilization can be obtained, if the whey is driedin an untreated state or if yeast, bred on the whey, is dried. In thiscase no waste water is obtained, if the vapours from the evaporator arenot considered.

If yeast is bred on whey (technical processes for the breeding of yeaston whey are reported by L. Forman et al. Kvasny prumysl, 21, 1975, Vol.12, pages 283-285) a biomass is obtained from a solution of lactose andacids, simple nitrogen compounds like ammonia and ammonium sulphatebeing added. Thus the protein content of the final product is increasedover the untreated material. The ash content of the product obtained byyeast breeding and drying of the total material is too high, as comparedwith the untreated product. This depends on the loss of weight at theyeast breading (ca 40% of the lactose) and on the sulphuric acid orammonium sulphate, that has been added to control the pH-value. (Theaddition of ammonium sulphate is equivalent to the addition of sulphuricacid, as the ammonia from the former is assimilated by the yeast and thesulphuric acid remains in solution). At the termination of the yeastbreeding, the acids are neutralized with alkali, which adds to the ashcontent. It is important to neutralize the acids, because otherwiseconcentrated acids will arise at the evaporation process. (Theconcentration ratio is about 500, as 1000 kgs of whey contain 940 kgs ofwater, and 40 kgs yeast with 4-5% water are obtained. Thus the finalproduct just contains 1.5-2 kgs of the original water content.)

It would be advantageous to add an acid, that is consumed during theyeast breeding step. According to the "Polyvit-process" nitric acid isused, that is utilized simultaneously as a source of nitrogen (B.Waeser, Chemiker Zeitung 7, 1944, pages 120-125). Today it is known,that in the case of oxygen deficiency, nitrite, formed from nitrate, mayreact spontaneously, or in the presence of microorganisms, withsecondary amines to form carcinogenous nitrous amines (J. SanderHoppe--Seyler's Z. Physiol. Chem. Vol. 349, 1968, pages 429-432). Anaddition of nitric acid cannot, consequently, be recommended. Lacticacid has proved to be advantageous for the control of the pH-value inaerobic yeast breading with mixed cultures of yeasts and lactic acidbacteria. (O. Moebus and P. Kiesbye, West German OS Specifications No.24 03 306, 24 10 349.5, 25 003 23.5).

The yeasts assimilate the lactic acid as a source of carbon. Lactic acidbacteria, bred together with the yeasts in a mixed culture, form lacticacid, that is needed for the pH-value control, even under aerobicconditions. Ammonia is added in controlled amounts to the whey and isbound to the lactic acid as ammonium lactate. Together with wheysolutions of carbo hydrates, starch or cellulose may generally be usedas substrate. In view of the expensive drying operation additionalconcentrated carbohydrate sources are added to the whey.

L. Forman et al. add ethanol to the whey and achieve a marked rise inthe dry solids. Such an addition can only be justified, if cheap,synthetic ethanol is available. If ethanol has to be produced from rawmaterials, containing starch, it is more convenient to decompose theseenzymatically and then use the products obtained together with the wheyfor the breeding of yeast (H. Muller, West German OS Specification No.25 48 641, filed Oct. 30, 1975). It would be necessary, however, to testthe cultures on the new medium. Until now, yeast strains that assimilatelactose, have been used for the breeding of yeast on whey, such asKluyueromyces fragilis (syn. Saccharomyces fragilis) and Candida kefir(syn. Candida pseudotropicalis). These strains cannot (according to J.Lodder, 1970, North Holland Publishing Company, Amsterdam, Holland),assimilate maltose. The culture yeasts, like Saccharomyces cerevisiaeare very well adapted to vegetable substrates, containing maltose. Itwould be convenient for this reason to use mixed cultures of yeasts andlactic acid bacteria with adapted assimilation characteristics for thebreeding of yeast on whey in combination with raw material, containingpolysaccharides.

It is an object of the present invention to develop an advantageousprocess of the type mentioned by way of introduction. It is also anobject of the present invention to provide a plant for the performanceof this process.

According to the present invention there is provided such a method,comprising a first process stage, in which the starch is converted todextrines, and the lactose is fermented to lactic acid by the action oflactic acid bacteria, that are able to ferment lactose and any othersugars or polysaccharides to acids, ammonia being added, whereupon, in asecond process stage the lactic acid suspension so obtained is used forthe breeding of yeast, additional ammonia and air being added, thedextrines being hydrolyzed by the action of amylases at the terminationof the process in the first stage, or alternatively during the processof the second stage.

In one advantageous embodiment of the present invention, starch isconverted to dextrines by the action of thermostable bacteria-a-amylase.

In another advantageous form of the present invention, Yoghourt-bacteriaLactobacillus bulgaricus and Streptococcus thermophilus are used for thefermentation.

It is preferred to breed Saccharomyces cerevisia in the second processstage, but other yeast strains may be used as well.

In one embodiment of the present invention, in which the dextrines arehydrolyzed during the second process stage, the amylases are addedcontinuously in order to prevent, to a great extent, any formation ofalcohol.

It is especially advantageous to perform the first process stage at atemperature, higher than 40° C., and to perform the second process stageat a temperature, that is not substantially higher than 32° C. In thefirst process stage lactic acid is produced by the action ofthermophilic lactic bacteria, and the temperature is set on such a highlevel, that the development of any detrimental microorganisms isprevented, the amylases utilized working at the same time at theirtemperature optimum. The temperature in the second process stage shouldbe low, to obtain a final product with a high content of yeast protein,and to obtain a high yield of yeast dry solids.

The second process stage of breeding yeast could be performed batchwise.It might be desirable, however, to perform the breeding of yeast as acontinuous operation. There are some problems, however, connected to anattempt to perform the yeast breeding on substrates of the type obtainedin the first process stage, according to the invention, in a continuousway in a steady state operation. If the rate of dilution, i.e. the ratioof the amount of liquid flow through a fermenter per unit of time to thevolume of liquid in the fermenter, is very high, the carbohydrates willpreferably be assimilated, whereby the concentration of lactic acid orthe lactate will rise in the culture. Finally, this concentration willbecome inhibiting to the yeast growth.

To overcome this disadvantage, the second process stage of the presentinvention is performed in a special semicontinuous way. Thus thebreeding of the yeast takes place in two distinct, repeated phases,phase I with a duration of time Δt₁ and phase II with a duration of timeΔt₂, suspension from the first process stage, containing lactice acid,ammonium lactate and carbo hydrates as sources for C and N being fed toa fermenter during the time Δt₁ in an amount of Δg. In phase II nofurther suspension is added, til the lactic acid, that has accumulatedin the culture solution during the time Δt₁ has been assimilated. At theend of phase II (after a period of time=Δt₂) an amount of yeastsuspension of weight Δg is withdrawn from the fermenter. The ratio ofΔg/Δt₁ is chosen in such a way, that Δt₂ >0.

The suspension may be fed, during phase I, in a steady flow, or adaptedto the yeast growth, e.g. according to a logarithmic pattern.

There are many ways of controlling the feed of suspension, such asmeasurement of the alcohol content in the fermenter, or by measuring theend point of the consumption of nutrition in phase II by means of anoxygen electrode, or carbondioxide electrode. The end point can as wellbe estimated by measurement of the paramagnetic characteristics ofresidual oxygen in the exhaust gas, or by measurement of the infraredabsorption of carbon dioxide in the exhaust gas. Further, the end pointcan be determined by measurement of the heat of reaction in thefermenter.

The process according to the invention is quite advantageous, as theaddition of raw material, containing starch, to the whey or otherresidual solutions with a lactose content, and performing the process,gives rise to final product with a reasonable ash content. There ispractically no waste water. The product obtained may be used as avaluable protein and vitamin source for feed. If culture yeasts arebred, the products may be used as food, as there are no toxicologicalobstacles for this.

The process according to the invention may be adapted to any availableraw materials. Apparatus for the performance of the process may bevaried in many ways in view of the desired final product.

Further features and advantages of the process and the apparatus forcarrying out the process will appear from the following description oftwo preferred embodiments thereof with reference to the accompanyingFIGURE, which is a schematic representation of a first embodiment ofapparatus for carrying out the process according to the invention.

The apparatus shown may be used in a variety of ways. The apparatuscomprises a tank 1 provided with heating and cooling means, an inlet 2for the lactose solution and an inlet 3 for starch containing rawmaterials, and a tank 4, provided with an inlet 5 for ammonia, and meansfor temperature control. Tank 1 is used for gelatinization andconverting of starch into dextrine, whereas tank 4 is intended for thelactic acid fermentation. The dextrine hydrolysis may be carried out inthis tank 4, too. Thus tank 1 and 4 are used for the first process stageof the invention. Further the apparatus comprises a tank 6 for the yeastbreeding, which tank 6 is provided with an ammonia inlet 7. Thus tank 6is used for the second process stage of the invention. The apparatuscomprises further items, that may be used according to the desired modeof operation.

Thus there are provided centrifugal separators 8 and 9 for theseparation of solids and lactic acid bacteria, and for preconcentrationof yeast suspension 10 from tank 6. Both separators are preferably ofthe selfemptying type. Also, there are means for the production of a dryfinal product, like an evaporator 11, a spray drier 12 and a baggingplant 13. An intermediate cooled tank 14 is provided for the storage offermented suspensions, yeast suspensions, centrifugal separationresidues and centrifugates. The different items are connected to eachother according to the lines drawn in the FIGURE.

EXAMPLE 1

In this example apparatus as shown in FIG. 1 was used. 4000 kgs ofwheatflour (63.3% starch, 13.3% raw protein/Kjeldahl-N×6.25/1.6% ashesand 11.6% water) were mixed at low temperature with 20,000 kgs of whey.An amount of thermostable bacteria-a-amylase (e.g. 900 mls Termamyl 60 LNOVO) sufficient for the conversion of starch into dextrines was added,and the mixture was heated to 75° C. by steam. (Gelatinization starts atabout 65° C., but the action of amylase makes the suspension lessviscous). The suspension was kept at 70°-80° C. for 30 minutes, and wasthen cooled to 50° C., whereupon 80 liters of yoghourt culture wasadded. The pH-value was adjusted to 6.2 by addition of 25% aqueousammonia and kept at 6.2 till 200 liters of 25% ammonia solution wereconsumed. After 10 hours of fermentation at 50° C. a pH-value of 4.3 wasachieved, that did not change noticeably after that. For the hydrolysisof the dextrines a fungal amylase (such as 500 mls of Fungamyl 800 LNOVO) is added. This can be done at the end of the lactic acidfermentation. It may also be convenient, however, to add the amylaseduring the yeast breeding, as the speed of reaction is sufficient evenat the lower temperature prevailing at this stage. In this case themaltose formation may be controlled, to minimize the alcohol formation.

EXAMPLE 2

In this example the yeast breeding was performed in a semicontinuousway. A suspension from the first process stage of the invention wasobtained with the following composition:

Lactid acid-D: 15.5 g/l

Lactic acid-L: 16.6 g/l

Ammonia: 4.5 g/l

Carbohydrates: 127 g/l (in the form of enzymatically decomposed starch).

The yeast breeding was performed in an aerated fermenter. In table 1 onephase I with continuous feed of substrate and one phase II without anyfeed are shown. Saccharomyces cerevisia was used.

From the table it is obvious, that lactic acid-C as well as lacticacid-L rise in concentration during phase I. The D-isomer of lactic acidis assimilated by the yeast at a higher rate than the L-form. After theO₂ -partial pressure has risen in the culture, i.e. at the end of phaseII, both isomers of lactic acid are practically completely assimilatedby the yeast.

                                      TABLE 1                                     __________________________________________________________________________    Yeast breeding on a substrate, obtained from whey + 20% wheat-flour,          by lactic acid fermentation and amylase-treatment, Temperature                = 23° C.                                                                          Lactic acid    COD (KMnO.sub.4)                                    Time                                                                              O.sub.2 -pressure                                                                    D g/l                                                                              L g/l  NH.sub.3                                                                         mg O.sub.2 /l                                       hours                                                                             % saturation                                                                         (cell-free solution)                                                                    pH                                                                              g/l                                                                              (cell-free solution)                                __________________________________________________________________________    0.00                                Start of feed                             0.50                                                                              55     0.43 0.67 4.8                                                                             0.39                                                                              320                                                1.50                                                                              15     0.77 1.21 4.8                                                                             0.54                                                                             2240                                                2.50                                                                              4      1.14 2.29 5.0                                                                             0.85                                                                             5120                                                3.50                                                                              3      1.57 3.54 5.1                                                                             1.05                                                                             5540      End of feed                               4.50                                                                              1      0.87 2.03 5.5                                                                             0.80                                                                             5120                                                6.50                                                                              1      0.17 0.15 6.0                                                                             0.37                                                                             4000                                                8.00                                                                              1      --   --   5.7                                                                             -- --                                                  8.75                                                                              55     0.07 0.12 5.8                                                                             0.25                                                                             1920      Recovery of yeast                         __________________________________________________________________________     Δg = 3 kgs                                                              Δt.sub.1 = 3.50 hours                                                   Δt.sub.2 = 5.25 hours                                                   Yeast growth/3 kgs substrate solution: 364 grams of yeast dry solids,         corr. to 1162 moist yeast substance.                                     

We claim:
 1. A process for producing yeast from dairy liquids containinglactose and natural materials containing sugars and polysaccharides,comprising in combination the steps of:a. preparing a mixture of thelactose containing dairy liquid and the natural materials containingsugars and polysaccharides, an amylase of the type that converts starchinto dextrine, and lactic acid bacteria; b. fermenting the lactose intolactic acid and converting the sugars and polysaccharides to dextrines,ammonia being added during this conversion to control the pH of themixture; c. subsequent to the lactose conversion step, adding amylase tohydrolyze the dextrines; and d. either concomitant with or subsequent tothe dextrine hydrolysis, adding a yeast to the suspension resulting fromthe lactose and sugar-polysaccharide and fermenting the mass underaerobic conditions, controlling the pH by addition of ammonia; e. andfurther wherein the yeast fermentation step (d.) is conductedsemicontinuously in two distinct repeated phases, as follows:(i) anamount, Δg, of suspension from the lactose and starch conversion stepcomprising lactic acid, ammonium lactate and carbohydrates as sourcesfor carbon and nitrogen is flowed to a fermentor during a time periodΔt₁, the lactic acid concentration increasing during Δt₁ to apredetermined level; (ii) after the lactic acid has reached saidpredetermined level, the flow of suspension from the lactose and starchconversion step is terminated for a time period Δt₂ to permit the lacticacid accumulated during the time period Δt₁ to become assimilated; (iii)at the termination of Δt₂, an amount of yeast suspension equal in weightto the suspension from the lactose and starch conversion step, Δg, iswithdrawn; (iv) thereafter the process steps (1)-(iii) are repeated. 2.The process of claim 1, wherein the diary liquid is whey and the naturalmaterials are selected from the group consisting of ground cereals andleguminous products.
 3. The process of claim 2, wherein the starch todextrine amylase is a thermostable bacteria alpha-amylase.
 4. Theprocess of claim 1, wherein the dextrine hydrolysis and yeastcultivation are carried out simultaneously.
 5. The process of claim 4,and further wherein the dairy liquid is whey and the natural materialsare selected from the group consisting of ground cereals and leguminousproducts.
 6. The process of claim 5, wherein the starch to dextrineamylase is a thermostable bacteria alpha-amylase.
 7. The process ofclaim 4, wherein the starch to dextrine amylase is a thermostablebacteria alpha-amylase.
 8. The process of claim 1, wherein the starch todextrine amylase is a thermostable bacteria alpha-amylase.
 9. Theprocess of claim 8, wherein the lactic acid bacteria is yogurt bacteriaLactobacillus bulgaricus and Streptococcus thermophilus.
 10. The processof claim 9, and further wherein the cultivated yeast is Saccharomycescerevisiae.
 11. The process of claim 1, wherein the amylase capable ofdextrine hydrolysis is added continuously throughout the yeastcultivation step to inhibit the formation of ethyl alcohol.
 12. Theprocess of claim 1, wherein the lactose conversion step is conducted ata temperature greater than 40° C. and the yeast cultivation step isconducted at a temperature not substantially higher than 32° C.